The present invention relates to improvements in or relating to Allium extracts. In particular, it relates to improvements in or relating to extending the therapeutic half-life or duration of Allium extracts. We describe a method of preparing an Allium species extract, the process comprising the steps of preparing a preparation of chopped, minced, ground or crushed cloves of an Allium species; allowing the preparation to stand for a first predetermined period at a first temperature; mixing with water at a second temperature for a second predetermined period; freezing the Allium/water mixture; maintaining the Allium/water mixture in a frozen state for a third predetermined period of time; allowing the Allium/water mixture to thaw at a third temperature; and removing solid material from the mixture to leave an aqueous Allium extract. We also describe a method of enhancing the methyl allyl-thiosulfinate and/or allyl methyl-thiosulfinate content of an aqueous Allium extract; the method comprising freezing the extract; allowing the frozen extract to begin to thaw; and collecting the liquid produced before thawing is complete; a method of enhancing the methyl allyl-thiosulfinate and/or allyl methyl-thiosulfinate content of an Allium species extract, the method comprising preparing a preparation of chopped, minced, ground or crushed cloves in an Allium species; adding methiin or a source of methiin to the Allium species preparation; and a method of extending the shelf-life or the bioactivity duration of an Allium species extract; the method comprising increasing a methyl allyl-thiosulfinate and/or allyl methyl-thiosulfinate content of the extract. We also describe an Allium species extract comprising allicin and at least one of methyl allyl-thiosulfinate and allyl methyl-thiosulfinate wherein the methyl allyl-thiosulfinate and allyl methyl-thiosulfinate is present in a combined amount of 17.5 wt % or more based on the combined amount of allicin, methyl allyl-thiosulfinate and allyl methyl-thiosulfinate, preferably 30 wt % or more.
S-Alk(enyl)-cysteine sulfoxides are widely distributed throughout the species in the genus Allium. Common examples of edible species that help form the approximate 700 species that belong to the genus Allium include: shallot (A. asacalonicum aust.), scallion (A. fitstulosum L.), leek (A. porrum L.), garlic (A. sativum L.), onion (A. cepa L.), chive (A. schoenoprasum L.), wild garlic (A. ursinum L.), Welsh onion (A. fistulosum L.), and Chinese chives (A. tuberosum L.).
Common to all Allium species is the enzyme allinase which catalyses the hydrolysis of S-alk(en)yl-L-cysteine sulfoxides (SACSs) in the presence of the cofactor pyridoxal 5′-phosphate to produce pyruvate, ammonia and sulfenic acids. In intact Allium species tissues, allinase is separately compartmentalized within plant vacuoles and the representative SACSs are located in the cytoplasm. Once Allium species tissue is damaged, the contents of the vacuole and cytoplasm mix, resulting in allinase being able to act on SACSs in a hydrolysis reaction leading to the formation of highly reactive sulfenic acids.
The sulfenic acids once formed, typically react with each other rapidly eliminating water in a condensation reaction that results in the synthesis of mixtures of thiosulfinates. The general reaction scheme showing the allinase-mediated hydrolysis of SACSs leading to the formation of sulfenic acids that can undergo condensation reaction with one another to form thiosulfinates is given in Scheme 1:

According to a comprehensive review article, [P. Rose, M. Whiteman, P. K. Moore and Y. Z. Zhu, Natural Product Reports, (2005), 22, pp. 351-368] concerning the biosynthesis, bioactivity and chemistry of SACSs, to date four major and two minor SACSs have been identified in the genus Allium and from these six SACSs approximately fifty additional sulfur containing compounds can be generated. The structures of the six SACSs commonly found in Allium species are given in Table 1:
TABLE 1Structures of six of the most common S-Alk(en)yl cysteine sulfoxides found in the genus Allium.Common Name Chemical Name Chemical StructureMethiin(+)-S-Methyl-L-cysteine sulfoxide  Ethiin(+)-S-Ethyl-L-cysteine sulfoxide Alliin(+)-S-Allyl-L-cysteine sulfoxide  Isoalliin(+)-S-Propenyl-L-cysteine sulfoxide Propiin (+)-S-Propyl-L-cysteine sulfoxide  Butiin(+)-S-n-Butyl-L-cysteine sulfoxide 
Condensation of two molecules of sulfenic acids gives rise to the formation of one thiosulfinate molecule (general formula RS(O)SR1 where R and R1 are alkyl and/or alkenyl groups). The thiosulfinates thus formed can be divided into two groups: (1) symmetrical thiosulfinates formed by the condensation of two molecules of sulfenic acid each with the same alk(en)yl substituent group R═R1 or (2) asymmetrical thiosulfinates generated from two different molecules of sulfenic acid each with a different alk(en)yl substituent (R≠R1). Thus, the thiosulfinate allicin [CH2CH═CH2S(O)SCH2CH═CH2, whose structure can be represented as AllS(O)SAll where All represents an allyl substituent] is a symmetrical thiosulfinate since R═R1═—CH2CH═CH2.
Since the six SACSs shown in Table 1 produce six different sulfenic acids due to hydrolysis brought about by allinase then numerous thiosulfinates can be produced. The structures of commonly produced thiosulfinates brought about by condensation reaction between the sulfenic acids that Allium species commonly produce have been identified [C. Shen, H. Xiao and K. L. Parkin, Journal of Agricultural and Food Chemistry, (2002), 50, pp. 2644-2651] and include those structures given in Table 2:
TABLE 2Structures of symmetric and examples of asymmetric thiosulfinates produced by the species in the genus Allium. Some thiosulfinates that contain a propenyl group(—CH═CH—CH3) also exist in the form of E,Z-geometric isomers. Alk(en)yl Groups In SymmetricalThiosulfinate RS(O)SR1ThiosulfinatesAlk(en)yl Groups In Asymmetrical ThiosulfinatesR = R1= —CH3R = —CH3 with R1 = —CH2—CH═CH2—CH2—CH3R = —CH3 with R1 = —CH2—CH2—CH3—CH2—CH2—CH3R = —CH3 with R1 = —CH═CH—CH3—CH2—CH═CH2R = —CH2—CH═CH2 with R1 = —CH3—CH═CH—CH3R = —CH2—CH═CH2 with R1 = —CH2—CH═CH2—CH2—CH2—CH2—CH3R = —CH2—CH═CH2 with R1 = —CH═CH—CH3R = —CH2—CH2—CH3 with R1 = —CH3R = —CH2—CH2—CH3 with R1 = —CH═CH—CH3R = —CH═CH—CH3 with R1 = —CH3R = —CH═CH—CH3 with R1 = —CH2—CH2—CH3
The pathways of thiosulfinate production have been studied [C. Shen, Z. Hong and K. L. Parkin, Journal of Agricultural and Food Chemistry, (2002), 50, pp. 2652-2659; and C. Shen and K. L. Parkin, Journal of Agricultural and Food Chemistry, (2000), 48, pp. 6254-6260] stemming from which it has been revealed that the precursor SACSs undergo hydrolysis at different rates. The reported rates of allinase mediated SACSs hydrolysis to form sulfenic acids are: (+)-S-trans-1-propenyl-L-cysteine sulfoxide (isoalliin)>(+)-S-allyl-L-cysteine sulfoxide (alliin)>(+)-propyl-L-cysteine sulfoxide (propiin)>(+)-S-ethyl-L-cysteine sulfoxide (ethiin)>(+)-S-methyl-L-cysteine sulfoxide (methiin). It is also reported that those thiosulfinates more rapidly formed from the SACSs that more rapidly undergo hydrolysis in an allinase mediated reaction can in turn react with the sulfenic acids produced by SACSs that are more slowly hydrolysed by the action allinase.
Allicin is known to have a range of therapeutic effects. However, its half-life is short [H. Fujisawa, K. Suma, K. Origuchi, T. Seki and T. Ariga, Journal of Agricultural and Food Chemistry, (2008), 56, pp. 4229-4235; and H. Fujisawa, K. Suma, K. Origuchi, T. Seki and T. Ariga, Bioscience, Biotechnology and Biochemistry, (2008), 72, pp. 2877-2883].
Although the vast majority of published scientific literature concerning the therapeutic action of garlic tends to focus on allicin [whose structural formula is: H2═CH—CH2—S(O)—S—CH2—CH═CH2 and whose abbreviation is AllS(O)SAll], there are a limited number of reports that indicate that both allyl methyl-thiosulfinate [whose structural formula is: CH3—S(O)—S—CH2—CH═CH2 and whose abbreviation is MeS(O)SAll] and methyl allyl-thiosulfinate [whose structural formula is: CH2═CH—CH2—S(O)—S—CH3 and whose abbreviation is AllS(O)SMe], also possess significant therapeutic biological activity. Examples of such reports are: (i) the potency of the antibacterial and antifungicidal activity of AllS(O)SMe tested against a range of gram-positive bacteria, gram-negative bacteria and yeasts has been found to be similar [H. Yoshida, H. Katsuzaki, R. Ohta, K. Ishikawa, H. Fukuda, T. Fujino and A. Suzuki, Bioscience, Biotechnology and Biochemistry, (1999), 63, pp. 591-594] to that of allicin; (ii) in vitro virucidal studies concerning compounds present in garlic extracts against a range of selected viruses including herpes simplex virus types 1 and 2, parainfluenza virus type 3 and rhinovirus type 2 have established that allicin, MeS(O)SAll and AllS(O)SMe thiosulfinates all possess virucidal activity; and (iii) that allicin, MeS(O)SAll and AllS(O)SMe all show inhibition [P. Canizares. I. Gracia, L. A. Gomez, C. M. de Argila, D. Boixeda, A. Garcia and L. de Rafael, Biotechnology Progress, (2004), pp. 397-401] of the in vitro growth of Helicobacter pylori. 
Hence, apart from the presence of allicin, it is advantageous for both the MeS(O)SAll and AllS(O)SMe thiosulfinate analogues to be present to support and help compliment the therapeutic activity of allicin in Allium species extracts, in particular those extracts derived from garlic. However, Block et al. [J. Agric. Food Chem., (1992), 40, pp. 2418-2430] have reported very significant variation in the quantities of MeS(O)SAll and AllS(O)SMe in extracts derived from different species of garlic obtained from different geographical locations and/or stored under different conditions prior to extraction. Some garlic extracts provide relatively very low concentrations of AllS(O)SMe and MeS(O)SAll. The present invention seeks to address this problem.
In view of allicin's relatively short half-life there is a need to enhance the therapeutic half-life of garlic and other Allium species extract compositions to overcome the limitations of the short half-life of allicin. The present inventions seek to provide a solution.